Modified nucleotides and oligonucleotides have gained an important role in the development of pharmaceuticals over the last several years. For example, analogs of nucleosides and nucleotides have been employed as antiviral compounds. Oligonucleotides comprised of nucleotide analog building blocks have been used as inhibitors of gene translation. (See, Huryn and Okabe (1992) Chem. Rev. 92:1745-1788).
The recent discovery of oligonucleotide library screening technology has opened up an additional area for the pharmaceutical application of nucleotide analogs in oligonucleotides; as highly specific, high affinity inhibitors of protein function. See, e.g., U.S. Pat. No. 5,270,163 entitled, Nucleic Acid Ligands; and Tuerk and Gold (1990) Science 249:505-510. This technology is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential Enrichment. SELEX can be carried out with libraries comprised of modified oligonucleotides to give ligands incorporating desired chemical functionalities (See, U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993 and entitled "High Affinity Nucleic Acid Ligands containing Modified Nucleotides").
Stability against nuclease degradation is a concern in the field of oligonucleotide therapeutics. Oligodeoxynucleotides are often stabilized by the introduction of phosphorothioate internucleotidic linkages. (See, Huryn and Okabe (1992) Chem. Rev. 92:1745-1788; Englisch and Gauss (1991) Angew. Chem. 30:613-722).
Modification of the 2'-position of pyrimidines has also been shown to stabilize oligonucleotides against nuclease degradation. (See, Paolella et al. (1992) EMBO Journal 11:1913-1919; Pieken et al. (1991) Science 253:314-317.) Both 2'-amino and 2'-fluoro nucleotides have been used for this purpose. The 5'-triphosphate derivatives of these modified nucleotides are substrates for T7 RNA polymerase. (Aurup et al. (1992) Biochemistry 31:9636-9641.)
The introduction of modifications to the 2'-position of pyrimidine nucleosides is not a highly efficient process. Furthermore, current technology allows only for the preparation of a few select 2'-modified pyrimidines under harsh reaction conditions with low yield. (Verheyden et al. (1971) J. Org. Chem. 36:250-254.) A general reaction allowing facile preparation of a wide variety of novel and known 2'-modified pyrimidines has to date not been available.
To facilitate incorporation into oligonucleotide libraries, nucleotide analogs have to be prepared as the 5'-triphosphate derivatives. This is the form that is recognized as a substrate for DNA dependent RNA polymerases. Furthermore, analogs also have to be prepared as the phosphoramidites in order to be incorporated into the final oligonucleotide ligand by automated chemical synthesis.
There currently is no reliable method for the stereoselective preparation of ribo-2'-hydroxylaminopyrimidines. Derivatives of such compounds have not been described nor have these compounds been characterized. It has been reported that the BH.sub.3 reduction of the oxime derivative of 2'-ketouridine affords mostly the 2'-hydroxylaminonucleosides of the arabino configuration. (Tronchet et al. (1990) Tetrahedron Lett. 31:531.) 2'-Halomethylpyrimidines are unknown. The 2'-aminopyrimidines are known compounds, however, they have never been prepared by an intramolecular introduction of the amino group. All previous procedures for synthesizing such compounds have proceeded through the 2'-azido precursor. (See, Verheyden et al. (1971) J. Org. Chem. 36:250-254.)
Cyclization reactions where a neighboring hydroxyl group is exploited as an anchor for a nucleophile which is then positioned to undergo cyclization with concomitant opening of an existing heterocycle have been observed in the opening of epoxyalcohols. (Jung and Jung (1989) Tetrahedron Lett. 30:6637-6640.) ##STR1## Oxidation and hydrolysis of B gave the desired .beta.-hydroxy .varies.-amino acid C. It is not obvious that the crucial cyclization step should work analogously to open 2,2'anhydropyrimidines. Furthermore, the reported hydrolysis conditions are incompatible with nucleosides.
Other examples of similar intramolecular openings of epoxides have been reported. Roush et al. showed that a phenylcarbamate group can be directed to open an adjacent epoxide through the carbamate nitrogen or oxygen, depending on the reaction conditions. (See, Roush et al. (1983) J. Org. Chem. 48:5093; Roush and Adam (1985) J. Org. Chem. 50:3752.) Many additional examples of intramolecular nucleophilic epoxy alcohol ring openings by carbon, nitrogen, oxygen, and sulfur nucleophiles, as well as reductive openings and chelated additions by organometallic species have been reported. (See, Intramolecular carbon nucleophilic openings of epoxy alcohols: McCombie et al. (1985) Tetrahedron Lett. 26:6301; McCombie et al. (1989) Tetrahedron Lett. 30:7029. Bicyclic products (e.g.; epoxides of cyclic olefins) Padwa et al. (1991) J. Org. Chem. 56:3556. Epoxy alcohol conversions by organometallic reagents: Me.sub.2 CuLi and/or Me.sub.3 Al: Roush et al. (1983) Tetrahedron Lett. 24:1377; Nishikubo and Kishi, (1981) Tetrahedron 37:3873; Johnson et al. (1979) Tetrahedron Lett. 20:4343. Ti(OiPr).sub.4 -Mediated: Caron and Sharpless, (1985) J. Org. Chem. 50:1557. Reductive openings of epoxy alcohols: Red-Al/DiBAl: Finan and Kishi, (1982) Tetrahedron Lett. 23:2719; Viti (1982) Tetrahedron Lett. 23:4541; Katsuki et al. (1982) J. Org. Chem. 47:1378; Nicolaou and Uenishi (1982) J. Chem. Soc. Chem. Common. 1292. Intramolecular nitrogen nucleophilic openings of epoxy alcohols: N-Hydroxyl amides: Roush and Follows (1994) Tetrahedron Lett. 35:4935. Alkyl and acyl carbamates: Knapp et al. (1987) Tetrahedron Lett. 28:5399; McCombie and Nagabhushan, ibid 5395; Roush and Adam (1985) J. Org. Chem. 50:3752; Roush and Brown, ibid, (1982) 47:1371; Minami et al. (1982) J. Amer. Chem Soc. 104:1109. Bicyclic products (e.g.; epoxides of cyclic olefins): Schubert, et al. (1986) Liebigs Annalen der Chemie 2009. Intramolecular oxygen nucleophilic openings of epoxy alcohols: McCombie and Metz (1987) Tetrahedron Lett. 28:383; Roush et al. (1983) J. Org. Chem. 48:5093; Katsuki et al. (1982) J. Org. Chem. 47:1378. The opening of an epoxide by an adjacent trichloroacetimidate has been reported: Bernet and Vasella, (1983) Tet. Letters 24:5491-5494.
2'-O-Methyl ethers of nucleosides are known to occur in nature as minor components of transfer RNA. (R. H. Hall, "The Modified Nucleosides in Nucleic Acids." Columbia University Press, New York, N.Y., 1971). 2'-O-Alkyl substituted nucleosides have been used to stabilize oligonucleotides against chemical and enzymatic degradation (For example see E. DeClerq et al., FEBS Letters, 1972, 24, 137; H. Inoue et. al., FEBS Letters, 1987, 215, 327; A. M. Iribarren et. al. , Proc. Natl. Acad. Sci. USA 1990, 87, 7747; G. Kawai et. al., Biochemistry 1992, 31, 1040.). 2'-O-Alkyl substitutents also can serve as removable protecting groups for the 2'-hydroxyl of ribonucleosides in oligonucleotide synthesis. (For example see K. Kikugawa et. al., Chem. Pharm. Bull. 1967, 16, 1110; H. Takaku et. al., J. Org. Chem. 1984, 49, 51; L. W. McLaughlin et. al., Synthesis, 1985, 322.).
2'-O-Alkyl nucleosides have been prepared by stannous chloride catalyzed reaction of free nucleosides and diazomethane followed by a tedious separation of alkylated isomers as described by M. J. Robins et. al., J. Org. Chem. 1974, 39, 1891. A further alkylation preceedure is described by D. Wagner et. al., J. Org. Chem. 1974, 39, 24. Where the free nucleosides uridine, cytidine and adenosine are alkylated by the reaction of a preformed 2',3'-O-dibutylstannylene nucleoside with alkyl halides to afford a mixture of 2'-O and 3'-O-alkylated products. Alternatively 2'-O-alkylated nucleosides have been obtained as the result of an exhaustive protection scheme of both the sugar and/or the heterocycle followed by selective alkylation of the free hydroxyl and removal of all the protecting groups. Notable is the use of the 5',3'-O-(tetraisopropyl-disiloxane) as described by H. Inoue et. al., Nucleic Acids Res. 1987, 15, 6131., V. A. Gladkaya et. al., Khim. Prir. Soedin, 1989, 4, 568; B. S. Sproat et. al., Nucleic Acids Res. 1989, 18, 41; T. Akiyama et. al., Bull. Chem. Soc. Jpn. 1990, 63, 3356. as well as tritylation for this purpose in Y. Furukawa et. al., Chem. Pharm. Bull. 1965, 13, 1273; E. Wagner et. al., Nucleic Acids Res. 1991, 19, 5965; K. Yamana et. al. , Tet. Let. 1991, 32, 6347. 2,2'-Anhydropyrimidines of some common nucleosides are commercially available (Aldrich: anhydrouridine, anhydrocytidine) or are easily prepared by those skilled in the art (for example see K. K. Ogilvie et al., Can. J. Chem. 1969, 47,495; A. Hampton et. al., Biochemistry, 1966, 5, 2076.). 8,2'-Anhydropurines are easily prepared by those skilled in the art (For a Review of methods see J. G. Moffatt in "Nucleoside Analogues", R. T. Walker et. al., Eds., Plenum Publishing Corp. 1979). K. K. Ogilvie et al. (1972) Con. J. Chem. 50:2249
Metal alkoxides are commercially available and methods of preparation are known to those skilled in the art. Meant as an example but not limited to the following see Gelest Inc., Tullytown, Pa., 1994-95 cataloque; Johnson Matthey, ALPHA, Ward Hill, Pa., 1994-95 catalogue, Aldrich Inc. catalogue. All metal alkoxides listed are included herein by reference. They are easily made by reaction of the metal with an excess of alcohol with optional heating and activation of the metal (ie. I.sub.2, HgX.sub.2), or reaction of organometalic compounds with alcohols, or metal hydrides with alcohols, or metal halides with alcohols or alkoxides (Na, K, other monovalent cation salts) or alcoholysis of a metal alkoxide with an excess of a second alcohol.